PROJECT SUMMARY In this project we will develop new technology for non-invasive and continuous therapeutic drug monitoring. Drug dosing is normally prescribed based on population averages, but in most cases direct clinical testing of systemic drug levels is performed infrequently or not at all. This is particularly problematic for drugs with narrow therapeutic indices, where treatment can be ineffective or outright toxic. Therefore, there is a persistent need for new technology for routine drug monitoring to allow better therapeutic outcomes while minimizing side-effects. We propose to address this problem by developing drug-sensitive fluorescent nanosensors that will use circulating red blood cell (RBC) ghosts as a vehicle to remain in circulations. By using near- infrared fluorophores at high local concentrations, these will produce drug-dependent signals that will be measurable with an external optical reader. Because unmodified RBCs are known to stay in circulation for weeks or months, this will allow long-term, continuous monitoring directly in the peripheral blood. Although there are many potential uses for this technology, we will first develop it for monitoring lithium and sodium as examples of a prescribed drug and its toxic side-effect. The project has three main phases. First we will design fluorescent red blood cell (f-RBC) nanosensors that circulate stably in the blood stream. These will encapsulate novel fluorescent sensor constructs for accurate quantification of Lithium and sodium blood concentrations. Second, we will develop an f-RBC fluorescence reader that for non-invasive and accurate quantification of f-RBC signals in vivo without having to draw blood samples. The reader will measure circulating f-RBC sensors in major blood vessels in the forearm in diffuse reflectance configuration. Third, we will validate and optimize our f-RBC sensor and reader in optical flow-phantom models in vitro and in rats treated with lithium and a diuretic in vivo. Longer term, we anticipate that there will be many uses for our f-RBC nanosensor technology for personalized therapeutic dose monitoring in many areas of medicine. The technology could also be extended to monitor effects on downstream drug targets in the future.